Entropic trapping and sieving of molecules

ABSTRACT

Nanofluidic entropic traps, comprising alternating thin and thick regions, sieve small molecules such as DNA or protein polymers and other molecules. The thick region is comparable or substantially larger than the molecule to be separated, while the thin region is substantially smaller than the size of the molecules to be separated. Due to the molecular size dependence of the entropic trapping effect, separation of molecules may be achieved. In addition, entropic traps are used to collect, trap and control many molecules in the nanofluidic channel. A fabrication method is disclosed to provide an efficient way to make nanofluidic constrictions in any fluidic devices.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/137,146, filed Jun. 1, 1999, the disclosure of whichis hereby incorporated herein by reference.

[0002] This invention was made with Government support under Grant no.HG01506, awarded by NIH. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates, in general, to the efficientseparation of molecules such as DNA and proteins, and more particularlyto a separation device including nanofluidic channels of different sizesfor providing alternate thin and thick regions along a channel to act asa filtering or sieving structure.

[0004] The separation of molecules according to their sizes is anessential step in biology and other fields and in analytical proceduressuch as chromatography, DNA sequencing or genome mapping. Conventionalmethods for separating molecules include electrophoresis andchromatography, which utilize the different transport properties(mobility) of different molecules in a solution-filled capillary orcolumn. In many cases, additional sieving material, such as a gelmatrix, is required to obtain sufficient separation of the molecules topermit analysis. In a conventional gel electrophoresis, as an example,molecules such as DNA molecules are separated during anelectric-field-driven motion in a highly restrictive gel matrix, becausethe mobility of the molecules is dependent on their length. However,this length-dependence of molecule mobility vanishes for DNA moleculeslonger than about 40,000 base pairs, mainly because the molecules tendto be more stretched and oriented in the direction of the appliedelectric field. Molecules as long as 10,000,000 base pairs can beseparated by pulsing the electric field (pulsed field gelelectrophoresis), but this process is usually very time consuming andinefficient.

[0005] To obtain better efficiency and control for separation process,the use of an artificial system using a precisely defined microchannelstructure as a molecular sieve has been suggested. However, initialattempts to produce efficient artificial gel systems were hindered bypoor understanding of the molecular dynamics in the microchannels. Ithas been found that the conformation (shape) of DNA or other polymermolecules has a direct impact on their motion in a restrictive mediumbecause the interaction cross section of the molecules with obstacles ischanged with conformational change. In free solution, polymer moleculessuch as DNA have a spherical shape in their equilibrium state, and thesize of this equilibrium shape is characterized by a radius of gyration(R_(o)) of the molecule. In the separation process of DNA or otherpolymers, it is important to maintain the conformation of the moleculein its equilibrium shape as much as possible, because otherwise thepolymeric molecule will stretch out in the direction of the motion,rendering the mobility of the molecule length (size) independent. Thisis because there is minimal difference in their interaction with aretarding matrix such as gel or obstacles.

[0006] In terms of the fabrication of artificial gel systems, currentphotolithography techniques are limited in resolution at about the 1micrometer level. Therefore, one cannot easily make constrictions orobstacles small enough for the separation of important molecules (DNA,proteins etc). Electron beam lithography can fabricate smaller featuresbut it generally is too expensive, and it is difficult to produce alarge-area device with this process.

[0007] It became clear that a more careful design of a separationdevice, combined with an inexpensive technique that can produce manyultrasmall constrictions over a large area, is essential in developing afunctioning molecular separation device.

SUMMARY OF THE INVENTION

[0008] When molecules become relaxed or are in their equilibriumspherical shape, their interaction with a retarding matrix can bedependent on the molecule's radius of gyration (R_(o)), and in turn onthe length of the molecule. Accordingly, a design for a molecule sievingstructure should include a somewhat open area where molecules can relax,as well as narrow constrictions that can serve as a molecular sieve.

[0009] It is, therefore, an object of the present invention to provide aseparation device incorporating nanofluidic constrictions (thin regions)and obstacle free regions (thick regions), through which molecules canbe caused to flow either by electrophoresis or by non-electric forces.

[0010] Briefly, the device of the invention provides a flow channelincorporating alternating thin and thick regions which operate as afilter, or sieving structure. The thin regions are sufficiently small toact as constrictions to the flow of small objects, such as DNAmolecules, proteins, cells, viruses, or other similarly-sized particles,while the thick regions allow molecules to relax for more efficientseparation at the thin region. To this end, the thick region depth maybe made comparable to, or substantially larger than, the size of amolecule (for example, the radius of gyration R_(o) for polymermolecules) to be sieved. Also the thin region depth may be madesubstantially smaller than the size of the molecule or other object tobe sieved. Although the device of the invention can be used to filter avariety of objects, the following description will be in terms ofmolecules, and particularly DNA molecules for convenience.

[0011] Accordingly, the present invention is directed to a nanofluidicchannel in which the motion of molecules such as DNA molecules ischaracterized by the provision of molecular traps. In accordance withthe invention, an elongated nanofluidic channel is provided withalternating regions of thick and thin gaps along its length. Theequilibrium spherical shape of a molecule such as DNA or protein has aradius of gyration R_(o), which is the shape the molecule assumes whenit is relaxed in an open region, such as in the thick regions of thechannel. If the molecule is forced to enter a constriction that is muchless than R_(o), the molecule has to be deformed from its equilibriumshape. Since such a deformation is entropically unfavorable, a drivingforce is required to force the molecule to enter the constriction. Thiseffect is referred to as the entropic trapping of a long polymer, andthis effect is crucial in the operation of present invention.

[0012] The entropic trapping effect can be utilized in operations suchas molecular trapping, molecular band formation, molecular separationand sieving, and molecular flow manipulation in nanofluidic ormicrofluidic channels. The separation or sieving can be achieved when asuitable driving force is supplied to trapped molecules, when theymigrate across many molecular traps and get separated because of thesize-dependent trapping effect. Just before the migration through thethin regions, molecules are sieved by entropic trapping effect. Afterthe molecules pass through the thin region, they relax back to theirequilibrium shape quickly because of the existence of the thick regions.This process is repeated many times until the required separation isachieved. By controlling the driving force for the molecules, moleculartrapping and manipulation can be achieved with the same structure.

[0013] In accordance with one embodiment of the invention, a new methodwas used to fabricate a nanofluidic channel having narrow constrictions(thin regions), spaced along the length of the channel, with the depthof the thin regions ranging between about 10 nm and about 500 nm, andhaving relatively thick regions between adjacent constrictions, ofbetween about 0.5 micrometer and about 10 micrometer. Channels of theseapproximate dimensions may be referred to herein as nanofluidicchannels, or simply as nanochannels. In accordance with this method,channels with variable depths were defined and etched in a siliconsubstrate, or wafer, using two-level photolithography. After a thermaloxidation process, mainly for electrical isolation, the top surface ofthe device was covered with a thin transparent plate. This techniquepermitted easy fabrication of very narrow gaps or constrictions withoutthe need for e-beam lithography for the patterning of sub-micrometerfeatures. This process was accomplished by the use of differentialetching of two regions and the bonding of a capping layer.

BRIEF DESCRIPTION OF DRAWINGS

[0014] The foregoing and additional objects, features and advantages ofthe present invention will be apparent to those of skill in the art fromthe following detailed description of preferred embodiments thereof,taken in conjunction with the accompanying drawings, in which:

[0015]FIG. 1 is a diagrammatic cross sectional view of an entropictrapping nanochannel in accordance with the present invention;

[0016]FIG. 2 is a diagrammatic top plan view of an entropic trap, whichillustrates the separation mechanism;

[0017]FIGS. 3 through 7 diagrammatically illustrate the fabricationprocess for the nanofluidic sieving device of the present invention;

[0018]FIG. 8 illustrates one embodiment of the nanochannel of theinvention in combination with cathode and anode electrodes and twoloading reservoirs;

[0019]FIG. 9 graphically illustrates the mobility of two different DNAspecies versus electric field in a nanofluidic sieving channel;

[0020]FIG. 10 illustrates the collection of DNA, launching, separationand detection of DNA bands, in one preferred embodiment of theinvention; and

[0021]FIG. 11 is a diagrammatic top plan view of a multiple channelentropic trap device, where two different DNA samples can be loaded andseparated simultaneously.

DESCRIPTION OF PREFERRED EMBODIMENT

[0022] Turning now to a more detailed consideration of the presentinvention, there is illustrated in FIG. 1 a nanofluidic sieving device10 in accordance with the present invention. The device 10 includes asilicon wafer or substrate 12 in which is fabricated a nanofluidicchannel 14 having alternating thick regions 16 and thin regions 18 alongits length. The channel 14 preferably is covered by a transparent topplate 22 which is bonded to the substrate 12 along the edges of thechannel. The nanochannel 14 is filled with a buffer solution or otherliquid containing DNA molecules or other polymer molecules 20 to beseparated. It will be understood that any desired material such as glassor plastic may be used as the substrate 12, and as the transparentcoverplate 22, and any conventional bonding techniques can be used toseal the coverplate 22 to a particular substrate 12.

[0023] In the illustrated embodiment of the invention, which is specificfor DNA molecule separation by way of example, the thick regions 16 maybe between about 0.5 micrometers and 5 micrometers in depth, orthickness, while the thin regions may be between 50 and 200 mm in depth,or thickness. The thicknesses of the thick and thin regions can bevaried according to the size of the molecule 20 to be separated. Thethin region 18 thickness (defined as t_(S)) is substantially smallerthan the radius of gyration R_(o) of the DNA or other polymer molecule20 to be separated. The thick region 16 thickness (defined as t_(d)) iscompatible to R_(o) of a molecule 20 to be separated, and thus to atypical long DNA or other polymer molecule, allowing the molecule torelax to its equilibrium spherical shape in this region. Becausemolecules can relax in the thick regions 16, they are entropicallyhindered from entering the thin regions 18 of the channel. When amolecule 20 to be separated is driven through the nanochannel 14 by anelectric field or by hydrodynamic pressure, the motion of the molecule20 will be retarded whenever it reaches the thin regions 18.

[0024]FIG. 2 is a top view of the nanofluidic sieving channel 14 wheretwo different DNA molecules or polymers 20 a (smaller) and 20 b (larger)were driven toward the right-hand end of the channel. Both molecules 20a and 20 b are trapped at starting points 22 of the thin regions 18. Thelarger molecule 20 b has a wider contact area with the thin region 18,as compared with the smaller molecule 20 a (w_(a)<w_(b)), which makesthe larger molecule 20 b have a higher probability of escaping thetrapping point and progressing through the channel.

[0025] The length of the thin region 18 (defined as l_(s)) and thelength of the thick region 16 (defined as l_(d)) along the length ofelongated nanochannel 14 can be varied to accommodate molecules withdifferent R_(o) and length. Changing l_(d) changes the relaxation of themolecule after it escapes the thin region 18. As the size of themolecule 20 increases, l_(d) should be increased to accommodate theincreased relaxation time required for big molecules to relax back toequilibrium shape. In the illustrated embodiments, the nanofluidicchannel is 30 micrometer wide (W), although other widths can beprovided. It will be understood that any desired number of nanochannelswith any desired combination of values of l_(s), l_(d), W, t_(s), t_(d)may be provided on a wafer, or substrate.

[0026] As illustrated in FIGS. 3 through 7, nanofluidic channels such asthe channels 14 illustrated in FIGS. 1 and 2, may be fabricated on asilicon wafer 30 by a photolithography and reactive ion etchingtechnique. In an experimental fabrication of nanochannels in accordancewith the invention, as illustrated in FIG. 3, a channel 32 was definedon the top surface 33 of the wafer 30 by standard photolithography, andwas etched by a reactive ion etch (RIE), providing a channel having afloor 34. Thereafter, as illustrated in FIG. 4, a second level ofphotolithography and chlorine RIE etching with an oxide mask were usedto make spaced thick regions 36 within the channel 30. This etching stepwas performed in the floor 34 of the channel 32 (FIG. 3) to produce asecond, lower floor 37 in each of the thick regions, leaving in thechannel a series of parallel transverse barriers 38 spaced apart alongthe length of the channel 32 between the thick regions. The barriersform the ends of the thick regions of the channel (region 16 in FIG. 1)with the tops 34 of the barriers forming the thin regions. Thestructural parameters l_(s), l_(d), W, t_(s), t_(d) in FIG. 1 can beeasily varied during these first two lithography steps with a highprecision, and according to the specific needs of the device.

[0027] After completing the channel 32, a pair of loading/unloadingapertures 40 and 42 were fabricated at opposite ends of the channel bypotassium hydroxide (KOH) etch-through using a silicon nitride etchmask. One of the two apertures 40 and 42 may serve as an inlet for abuffer solution or other liquid, containing molecules to be separated,while the other aperture may serve as the outlet for the solution andthe separated molecules. Alternatively, the aperture need not befabricated, but the channel 32 may instead be connected to othermicrofluidic or nanofluidic channels or chambers that have differentfunctions, to form an integrated system.

[0028] As illustrated in FIG. 6, a thermal oxide layer 50 may be grownon all of the surface of the channel 32 and on the surface of the waferto a thickness of up to 400 nm to provide electrical isolation betweenthe buffer solution and the silicon substrate. In the case where anon-conducting substrate, or wafer 30, such as glass is used, this stepmay be omitted.

[0029] Finally, as illustrated in FIG. 7, the top of the channel 32 washermetically sealed with a thin glass coverslip 52 secured to the topsurface 54 of the silicon substrate 30 and its oxide coating 50, as byanodic bonding, to provide the nanofluidic channel 56. The coverslip 52may be a thin Pyrex glass or other suitable material to close thechannel and to provide a fluid path across the barriers 38 from theinlet end 40 to the outlet end 42. In the case of using substrates 30other than silicon, the hermetic seal may be obtained by suitablebonding techniques such as glass-to-glass fusion bonding or bonding withan intervening thin glue layer. The coverplate 52 is thin andtransparent enough to allow the detection of separated molecules.

[0030] In one preferred embodiment of a nanofluidic channel device, asillustrated in FIG. 8, the nanofluidic sieving device 56 illustrated inFIG. 7 is turned upside down, and two liquid reservoirs 58 and 60,respectively, are attached. Metallic wires 62 and 64, preferably noblemetals such as platinum or gold, may be inserted into the reservoirs 58and 60 respectively, to make a cathode 66 and an anode 68. A voltage Vapplied across the electrodes produces separation of molecules 20, whichis detected from the bottom side through the transparent coverplate 22of the device 10. The detection of molecules 20, as an example not as alimitation, may be done by using a fluorescent dye attached uniformly tothe molecules 20 and observing in the channel by an optical microscope70 or equivalent optical detection system.

[0031]FIG. 9 is a graphical illustration of the mobility of twodifferent (large and small) molecules versus the electric field appliedas a driving force to the nanofluidic sieving channel. The driving forcefor the molecules in the channel may also come from hydrodynamicpressure if desired, and in such a case the pressure will be therelevant quantity, instead of the electric field as given in thisexample. It is understood that the mobility curves plotted versus theelectric field have a sigmoidal shape as shown in FIG. 9. The curve 80for larger polymer molecule should be higher than the curve 82 for asmaller polymer in a particular range 84 of the electric field. Ifelectric field is higher (in the range 86), the mobility is the sameirrespective of the molecule size, because the driving force is toostrong and the entropic trapping is negligible. If the electric field islower (in the range 88), then the entropic trapping is so strong thatmolecules are trapped indefinitely, irrespective of their size. Theelectric field applied to the nanofluidic channel should be adjusted tothe level corresponding to the range 84. The specific value for thisrange may vary for a specific molecules to be separated. If the electricfield is adjusted to the range 86, all the molecules move at the samespeed, irrespective of the size. Therefore, this range 86 may be usedfor recollection of already separated molecules or moving the mixture ofDNA molecules from one location to another without fractionating them.The electric field range 88 allows molecules to be collected at thefirst entropic barrier, because in the range 88 the entropic trappingeffect is too severe for DNA to overcome even a single entropic barrierwithin a reasonable amount of time.

[0032] As illustrated in FIG. 10, by way of an example and notlimitation, if a number of molecules are supplied to channel 14, as byway of reservoir 60 and aperture 42, and an electric field in the range88 in FIG. 9 is applied for a specific amount of time along thenanofluidic sieving channel 14, one can collect many DNA or polymermolecules 20 at the first entropic trap 90, yielding a highly definedand concentrated molecule band 92. The concentrated band 92 may belaunched into the nanochannel for band separation by switching theelectric field from the value in the range 88 of FIG. 9 to the value inthe range 84 of FIG. 9. In this illustrated embodiment of the invention,two different types of DNA (20 a and 20 b, small and large DNA,respectively) are mixed in the band 92. When launched into thenanochannel, the band 92 becomes separated, as it migrates through manyentropic traps along the channel, into two bands, a first band 94 and asecond band 96. It is understood that the first band is composed oflarger DNA 20 b, while the second band is composed of smaller DNA 20 a.

[0033] For the detection of this separation, in one preferredembodiment, one may set up a region of interest 98 and collect thefluorescent signal from the bands 94 and 96, either optically or usingother suitable methods, as a function of time. The separated bands 94and 96, may then be recollected at the other end of the channelsequentially, preferably in aperture 40 and reservoir 58, or otherfluidics channels may be used to redirect each band into separatemicrofluidic chambers.

[0034] It is imperative to note that the above-mentioned method may beutilized to fractionate mixtures with any number of different types ofmolecules, as the resolution permits. The resolution may be improved byapplying several different optimization techniques. Having a longerchannel is one way, but another important method is changing the variousstructural parameters mentioned in FIG. 1 to get optimized results. Forcertain polymer molecules, one may optimize a specific set ofconditions, including but not limited to, the structural parametersillustrated in FIG. 1, the electric field or the electric field range 84of FIG. 10, and the overall length of the nanochannel.

[0035] As diagrammatically illustrated in the top plan view of FIG. 11,by way of example and not limitation, a multiple channel device 98,which is capable of separating multiple samples simultaneously, may befabricated. In this embodiment of the invention, several nanofluidicsieving channels 100, 102, 104 and 106, each with a different sievingstructural parameter, are connected to a larger loading and collectionchamber 108. The different structural parameters are optimized for theseparation of different length ranges of molecules to be separated. Thenumber of nanochannels which may be connected to a loading or collectionchamber 108 may be increased without any difficulty in the fabricationor operation of the device, mainly to accommodate wide variety ofmolecules. The loading and collection chamber 108 is connected to thecathode by a wider channel 110, and to a reservoir of sample solution bya loading channel 112. In addition, the central collection chamber 108is defined by two entropic barriers 114 and 116, which enablemanipulation of the molecules to be separated, which are in the centralcollection chamber 108. The central chamber 108, the loading channel 112and the channel 110 are all supported by a supporting pillar structure120, mainly to prevent possible collapse of the coverplate (roof) of thechannel down to the bottom.

[0036] In the embodiment of the invention illustrated in FIG. 11, thereare two multiple channel devices 98 and 98's, having two separateloading and collection chambers 108 connected to two separate samplereservoirs (sample reservoirs A and B). Each loading and collectionchamber 108 is connected to the same sets of nanofluidic sievingchannels 100, 102, 104 and 106, with various structural parameters, andeventually all of these nanochannels 100 lead to a common anode, whereasthe two loading chambers 108 also lead to a common cathode.

[0037] In the operation of the device of FIG. 11, two different samplesof molecules, possibly one unknown sample to be analyzed and one knowncontrol or reference sample with size information about the fragments inthe sample (in DNA analysis for example, a DNA ladder sample could serveas a reference) may be introduced into sample reservoirs. For loadingthe molecules into the channels, a suitable electrical potential may beapplied between the cathode and the sample reservoirs, causing themolecules to enter the loading channel 112, the central collectionchamber 108, and the channel to the cathode 110. As a result, thecentral chamber 108 would be evenly filled with molecules to beseparated. Then another electric field is applied between the cathodeand the anode, causing molecule transport to the nanofluidic sievingchannels 100, 102, 104 and 106. The electric field between the cathodeand the anode may be selected to have a value in the electric fieldrange 88 of FIG. 9, so the molecules are collected at the very firstbarriers of each nanochannel. With this low electric field, themolecules behind the entropic barrier 114 cannot drift into the centralcollection chamber 108, but pile up behind the barrier 114. Additionallythe existence of the barrier 116 makes sure that the molecules in theloading channel 112 do not drift into the central chamber 108 sincethere is no substantial electric field existing between the samplereservoir and the cathode. Therefore, only the molecules in thecollection chamber 108 can drift into the nanochannels, providing theconcentrated band discussed with respect to FIG. 10 which will belaunched into each of the nanochannels.

[0038] After this process, the field may again be developed between thecathode and the sample reservoirs, causing the remainder of themolecules behind the entropic barrier 114 to be drained back to thesample reservoir, without affecting the collected molecules at the firstbarriers of the nanochannels 100, 102, 104 and 106. This process permitscontrol of the concentration of molecules in the launching band, whichis relevant in the separation process. Also, the same process can berepeated as many times as desired, to obtain even higher concentrationsof the molecules in the band.

[0039] As the separation process proceeds, the data taken from differentsamples can be easily detected and compared, enabling more reliableanalysis. It is important to know that the number of samples to beanalyzed may be increased as desired without any serious technical andoperational difficulties.

[0040] Thus, there has been disclosed a nanofluidic channel for use inentropic trapping and sieving of polymer molecules such as DNA andproteins. The channel includes alternating thick and thin segments, orsections, which alternately cause DNA or other polymer molecules tostretch and to return to a rest equilibrium configuration. The channelpermits separation of long polymers in a DC applied electric field, withthe device structure affecting the mobility of the molecules as theypass through the channels. Entropic traps have other uses inmanipulating and collecting many molecules, with a high degree ofcontrol, into a narrow band, which is useful in the separation process.Although the invention has been disclosed in terms of preferredembodiments, it will be apparent that variations and modifications maybe made without departing from the true spirit and scope thereof as setforth in the following claims.

What is claimed is:
 1. A nanofluidic device comprising: at least onechannel including a combination of thin and thick regions, the thinregions in the channel being thin enough to act as a constriction ofsmall objects, and the thick regions in the channel having a thicknessgreater than that of said thin regions, and being large enough to allowthe passage of said objects.
 2. The device of claim 1, further includingchannels leading from said at least one channel to fluidic components.3. The device of claim 2, further including a force to drive saidobjects through said channel.
 4. The device of claim 3, furtherincluding inlet and outlet apertures for said channel.
 5. The device ofclaim 4, wherein a part of said combination of thin and thick regions isused to collect and trap said objects.
 6. The device of claim 5, whereinsaid thin regions are used to control the flow of said objects inmicrofluidic channels and chambers.
 7. The device of claim 6, wherein atleast some of said thin and thick regions contains supporting structuresto maintain the uniformity of the thickness of the said regions.
 8. Thedevice of claim 7, wherein said thin and thick regions are alternatelyspaced along said channel.
 9. The device of claim 8, wherein thethickness of said thin region is between about 5 nm and about 500 nm.10. The device of claim 9, wherein the thickness of said thick region isbetween about 0.1 micron and about 10 micron.
 11. The device of claim10, further including means for applying a voltage across said channelfor producing electrophoresis of said objects.
 12. The device of claim11, further including fluidic channels for introducing said objects intothe channel.
 13. The device of claim 12, further including multiplenanofluidic sieving channels, optimized for different size range, forreceiving a mixture of wide variety of objects.
 14. The device of claim13, further including a fluidic chamber, bounded by said regions forcollecting molecules into a narrow band for further analysis.
 15. Thedevice of claim 14, further including multiple loading and collectionchambers, which are separate from one another, each containing a set ofmultiple channels for the analysis of multiple samples.
 16. A method forseparating DNA molecules, proteins, cells or other similar-sizedobjects, comprising; supplying molecules in a fluid to a nanofluidicchannel having alternating thin and thick regions, the thin regionsbeing smaller than the size of the molecule to be separated, and thethick region being substantially close to or larger than the size of themolecule to be separated; and, applying a driving force to cause saidmolecules to move along said channel through said regions, the thinregions causing trapping of selected molecules.
 17. The method of claim16, further including changing the length of the thick regions to varythe relaxation property of the molecule in the thick regions.
 18. Themethod of claim 17, further including collecting many molecules into anarrow band, by way of applying an appropriate driving force andentropic barriers in the microfluidic or nanofluidic channels.
 19. Themethod of claim 19, further including launching the collected moleculesby suddenly increasing the applied driving force, enabling molecules toescape the barrier.
 20. A method for fabricating a nanofluidic device,comprising; forming a first pattern of shallow regions in a substrate;forming another pattern of deep regions, excluding regions to be made asthin regions; and covering the patterns of shallow and deep regions toform a nanofluidic device with thin and thick regions.
 21. The method ofclaim 20, wherein the steps of forming the first and second patternscomprises a two-level lithography and etching process.
 22. The method ofclaim 21, further including covering the first and second patterns ofshallow and deep regions with an electrically insulating layer.
 23. Themethod of claim 22, wherein the covering of the regions includes bondinga plate to the substrate, the plate extending over the shallow and deepregions.
 24. A device for separating molecules, the device comprising: aplurality of alternating constricted and unconstricted regions forming achannel; the unconstricted regions having a transverse dimension andlength sufficient to allow a larger molecule to approach its equilibriumshape as it moves through the channel in response to a driving force;and, the constricted regions having a transverse dimension sufficientlysmall to influence the shape of some of the molecules moving through thechannels.
 25. The device of claim 24 wherein the constricted regionsprovide a trapping point adjacent an unconstricted region, and whereinthe larger molecules have a wider contact area at the trapping point ofthe constricted regions, and thus have a higher probability of escapingthe unconstricted region through a constricted region than a smallermolecule.
 26. The device of claim 24 wherein molecules in theunconstricted regions are in a relaxed state, and are entropicallyhindered from entering adjacent constricted regions in the channel. 27.The device of claim 24 and further comprising a substrate supporting thechannel.
 28. The device of claim 24 wherein the constricted regions arenanofluidic, and the unconstricted regions are obstacle free.
 29. Thedevice of claim 24 wherein the equilibrium spherical shape of a smallermolecule has a radius of gyration, and wherein the constricted regionhas a transverse dimension less than such radius of gyration.
 30. Thedevice of claim 24 wherein both large and small molecule need to deformfrom their equilibrium states to enter the constricted region.
 31. Thedevice of claim 24 wherein the equilibrium shape of the larger moleculeis influenced by the constricted region to a greater extent than theequilibrium shape of a smaller molecule.
 32. A device for separatingmolecules, the device comprising: a plurality of alternating constrictedand unconstricted regions forming a channel; the unconstricted regionshaving a depth and length sufficient to allow a larger molecule toapproach its radius of gyration as it moves through the channel inresponse to a driving force; the constricted regions having a depth lessthan a radius of gyration of a smaller molecule; and means for applyingforce to molecules in the channel.
 33. The device of claim 32 whereinthe constricted regions provide a trapping point adjacent anunconstricted region, and wherein the larger molecules have a widercontact area at the trapping point of the constricted regions, and thushave a higher probability of escaping the unconstricted region through aconstricted region than a smaller molecule.
 34. The device of claim 32wherein molecules in the unconstricted regions are in a relaxed state,and are entropically hindered from entering adjacent constricted regionsin the channel.
 35. The device of claim 32 and further comprising asubstrate supporting the channel.
 36. The device of claim 32 wherein theconstricted regions are nanofluidic, and the unconstricted regions areobstacle free.
 37. The device of claim 32 wherein the equilibriumspherical shape of a smaller molecule has a radius of gyration, andwherein the constricted region has a transverse dimension less than suchradius of gyration.
 38. The device of claim 32 wherein both the largerand smaller molecule need to deform from their equilibrium states toenter the constricted region.
 39. A device for separating molecules, thedevice comprising: an input reservoir and an output reservoir; aplurality of alternating constricted and unconstricted regions forming achannel coupled between the input and output reservoir; theunconstricted regions having a depth and length sufficient to allow alarger molecule to approach its equilibrium spherical shape as it movesthrough the channel in response to a driving force; and, the constrictedregions having a depth less than an equilibrium spherical shape of asmaller molecule.
 40. The device of claim 39, wherein the input andoutput reservoirs are positioned to contain a buffer solution withmolecules to be separated.
 41. The device of claim 40 and furthercomprising a first contact positioned within the input reservoir tocontact the buffer solution and a second contact positioned within theoutput reservoir to contact the buffer solution.
 42. The device of claim39 and further comprising a detector positioned about the channel todetect desired molecules in the channel.
 43. The device of claim 42wherein the detector comprises an optical microscope.
 44. A device forseparating molecules, the device comprising: a loading chamber; aplurality of separation channels coupled to the loading chamber, eachseparation channel having a plurality of alternating constricted andunconstricted regions; the unconstricted regions having a depth andlength sufficient to allow a larger molecule to approach its equilibriumspherical shape as it moves through the separation channel in responseto a driving force; and, the constricted regions having a depth lessthan an equilibrium spherical shape of a smaller molecule.
 45. Thedevice of claim 44 wherein different separation channels have differentstructural parameters selected from the group consisting of a transversedimension and length of each of the regions.
 46. The device of claim 45wherein the parameters are optimized for the separation of differentlength ranges of molecules.
 47. The device of claim 44 wherein theloading chamber comprises multiple support pillars.
 48. The device ofclaim 44 wherein the loading chamber is coupled to a loading channel byan entropic barrier.
 49. The device of claim 44 wherein the loadingchamber is coupled to a first electrical contact through an entropicbarrier.
 50. The device of claim 49 wherein the separation channels arecoupled to a second electrical contact, and wherein the first and secondelectrical contacts provide an electric field for driving moleculesthrough the separation channels when coupled to a power source.
 51. Adevice for separating larger molecules from smaller molecules, thedevice comprising: a channel having a depth and length sufficient toallow larger molecules to approach their equilibrium spherical shape;and means for creating a series of entropic barriers to selectedmolecules in the channel.
 52. The device of claim 51 and furthercomprising means for driving the molecules through the channel.
 53. Adevice for separating molecules, the device comprising: a sequence of anunconstricted region and an entropic barrier forming a channel; theunconstricted region having a transverse dimension and length sufficientto allow selected molecules to approach their equilibrium shape as theymove through the channel in response to a driving force; and, theentropic barrier influencing the shape of selected molecules as theymove through the channel.
 54. The device of claim 53 wherein theentropic barrier provides a differential delay of molecules movingthrough the channel based on the size of the molecules.
 55. The deviceof claim 53 and further comprising further alternating unconstrictedregions and entropic barriers forming the channel.
 56. A device forseparating molecules, the device comprising: a plurality of alternatingconstricted and unconstricted regions forming a channel; theunconstricted regions having a transverse dimension and lengthsufficient to allow a larger molecule to approach its equilibrium shapeas it moves through the channel in response to a driving force; and, theconstricted regions having a transverse dimension sufficiently small tomodulate a time it takes selected molecules to pass through theconstricted regions, wherein both large and small molecules pass throughthe channel.